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Keskin, Derin Benerci

Broad coverage of the pathogen population is particularly important when designing CD8+ T-cell epitope vaccines against viral pathogens. Traditional approaches are based on combinations of highly conserved T-cell epitopes. Peptide block entropy analysis is a novel approach for assembling sets of broadly covering antigens. Since T-cell epitopes are recognized as peptides rather than individual residues, this method is based on calculating the information content of blocks of peptides from a multiple sequence alignment of homologous proteins rather than using the information content of individual residues. The block entropy analysis provides broad coverage of variant antigens. We applied the block entropy analysis method to the proteomes of the four serotypes of dengue virus (DENV) and found 1,551 blocks of 9-mer peptides, which cover 99% of available sequences with five or fewer unique peptides. In contrast, the benchmark study by Khan et al. (2008) resulted in 165 conserved 9-mer peptides. Many of the conserved blocks are located consecutively in the proteins. Connecting these blocks resulted in 78 conserved regions. Of the 1551 blocks of 9-mer peptides 110 comprised predicted HLA binder sets. In total, 457 subunit peptides that encompass the diversity of all sequenced DENV strains of which 333 are T-cell epitope candidates.

Persistent infection with high-risk human papilloma viruses (HPV) is the worldwide cause of many cancers, including cervical, anal, vulval, vaginal, penile, and oropharyngeal. Since T cells naturally eliminate the majority of chronic HPV infections by recognizing epitopes displayed on virally altered epithelium, we exploited Poisson detection mass spectrometry (MS(^3)) to identify those epitopes and inform future T cell-based vaccine design. Nine cervical cancer biopsies from HPV-16 positive HLA-A02 patients were obtained, histopathology determined, and E7 oncogene PCR-amplified from tumor DNA and sequenced. Conservation of E7 oncogene coding segments was found in all tumors. MS(^3) analysis of HLA-A02 immunoprecipitates detected E7({11–19}) peptide (YMLDLQPET) in seven of the nine tumor biopsies. The remaining two samples were E7({11–19}) negative and lacked the HLA-A02 binding GILT thioreductase peptide despite possessing binding-competent HLA-A02 alleles. Thus, the conserved E7({11–19}) peptide is a dominant HLA-A02 binding tumor antigen in HPV-16 transformed cervical squamous and adenocarcinomas. Findings that a minority of HLA-A02:01 tumors lack expression of both E7({11–19}) and a peptide from a thioreductase important in processing of cysteine-rich proteins like E7 underscore the value of physical detection, define a potential additional tumor escape mechanism and have implications for therapeutic cancer vaccine development.

Vaccine-elicited cytotoxic T lymphocytes (CTL) recognizing conserved fragments of a pathogen’s proteome could greatly impact infectious diseases and cancers. Enabling this potential are recent advances in mass spectrometry that identify specific target peptides among the myriad HLA-bound peptides on altered cells. Ultrasensitivity of these physical detection methods allows for the direct assessment of peptide presentation on small numbers of tissue-derived cells. In addition, concurrent advances in immunobiology suggest ways to induce CTLs with requisite functional avidity and tissue deployment. Elicitation of high-avidity resident-memory T cells through vaccination may shift the vaccinology paradigm both for preventive and therapeutic approaches to human disease control.

Human leukocyte antigens (HLA) are important biomarkers because multiple diseases, drug toxicity, and vaccine responses reveal strong HLA associations. Current clinical HLA typing is an elimination process requiring serial testing. We present an alternative in situ synthesized DNA-based microarray method that contains hundreds of thousands of probes representing a complete overlapping set covering 1,610 clinically relevant HLA class I alleles accompanied by computational tools for assigning HLA type to 4-digit resolution. Our proof-of-concept experiment included 21 blood samples, 18 cell lines, and multiple controls. The method is accurate, robust, and amenable to automation. Typing errors were restricted to homozygous samples or those with very closely related alleles from the same locus, but readily resolved by targeted DNA sequencing validation of flagged samples. High-throughput HLA typing technologies that are effective, yet inexpensive, can be used to analyze the world’s populations, benefiting both global public health and personalized health care.

BACKGROUND: The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing. RESULTS: When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development. CONCLUSIONS: The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues.

High-risk human papillomaviruses (HPVs) are the causes of many cancers, including cervical, anal, vulvar, vaginal, penile and oropharyngeal. To facilitate diagnosis, prognosis and characterization of these cancers, it is necessary to make full use of the immunological data on HPV available through publications, technical reports and databases. These data vary in granularity, quality and complexity. The extraction of knowledge from the vast amount of immunological data using data mining techniques remains a challenging task. To support integration of data and knowledge in virology and vaccinology, we developed a framework called KB-builder to streamline the development and deployment of web-accessible immunological knowledge systems. The framework consists of seven major functional modules, each facilitating a specific aspect of the knowledgebase construction process. Using KB-builder, we constructed the Human Papillomavirus T cell Antigen Database (HPVdb). It contains 2781 curated antigen entries of antigenic proteins derived from 18 genotypes of high-risk HPV and 18 genotypes of low-risk HPV. The HPVdb also catalogs 191 verified T cell epitopes and 45 verified human leukocyte antigen (HLA) ligands. Primary amino acid sequences of HPV antigens were collected and annotated from the UniProtKB. T cell epitopes and HLA ligands were collected from data mining of scientific literature and databases. The data were subject to extensive quality control (redundancy elimination, error detection and vocabulary consolidation). A set of computational tools for an in-depth analysis, such as sequence comparison using BLAST search, multiple alignments of antigens, classification of HPV types based on cancer risk, T cell epitope/HLA ligand visualization, T cell epitope/HLA ligand conservation analysis and sequence variability analysis, has been integrated within the HPVdb. Predicted Class I and Class II HLA binding peptides for 15 common HLA alleles are included in this database as putative targets. HPVdb is a knowledge-based system that integrates curated data and information with tailored analysis tools to facilitate data mining for HPV vaccinology and immunology. To our best knowledge, HPVdb is a unique data source providing a comprehensive list of HPV antigens and peptides. Database URL: http://cvc.dfci.harvard.edu/hpv/

Background: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. Results: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. Conclusions: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.

Introduction: Effective combination immunotherapeutic strategies may be required to enhance effector cells’ anti-tumor activities and improve clinical outcomes. Methods: XBP1 antigen-specific cytotoxic T lymphocytes (XBP1-CTL) generated using immunogenic heteroclitic XBP1 US184-192 (YISPWILAV) and XBP1 SP367-375 (YLFPQLISV) peptides or various solid tumor cells over-expressing XBP1 target antigen were evaluated, either alone or in combination with lenalidomide, for phenotype and immune functional activity. Results: Lenalidomide treatment of XBP1-CTL increased the proportion of CD45RO+ memory CD3+CD8+ T cells, but not the total CD3+CD8+ T cells. Lenalidomide upregulated critical T cell activation markers and costimulatory molecules (CD28, CD38, CD40L, CD69, ICOS), especially within the central memory CTL subset of XBP1-CTL, while decreasing TCRαβ and T cell checkpoint blockade (CTLA-4, PD-1). Lenalidomide increased the anti-tumor activities of XBP1-CTL memory subsets, which were associated with expression of Th1 transcriptional regulators (T-bet, Eomes) and Akt activation, thereby resulting in enhanced IFN-γ production, granzyme B upregulation and specific CD28/CD38-positive and CTLA-4/PD-1-negative cell proliferation. Conclusions: These studies suggest the potential benefit of lenalidomide treatment to boost anti-tumor activities of XBP1-specific CTL against a variety of solid tumors and enhance response to an XBP1-directing cancer vaccine regime.

Background: In this study, we used a systematic bioinformatics analysis approach to elucidate genes that exhibit an endothelial cell (EC) restricted expression pattern, and began to define their regulation, tissue distribution, and potential biological role. Results: Using a high throughput microarray platform, a primary set of 1,191 transcripts that are enriched in different primary ECs compared to non-ECs was identified (LCB >3, FDR <2%). Further refinement of this initial subset of transcripts, using published data, yielded 152 transcripts (representing 109 genes) with different degrees of EC-specificity. Several interesting patterns emerged among these genes: some were expressed in all ECs and several were restricted to microvascular ECs. Pathway analysis and gene ontology demonstrated that several of the identified genes are known to be involved in vasculature development, angiogenesis, and endothelial function (P < 0.01). These genes are enriched in cardiovascular diseases, hemorrhage and ischemia gene sets (P < 0.001). Most of the identified genes are ubiquitously expressed in many different tissues. Analysis of the proximal promoter revealed the enrichment of conserved binding sites for 26 different transcription factors and analysis of the untranslated regions suggests that a subset of the EC-restricted genes are targets of 15 microRNAs. While many of the identified genes are known for their regulatory role in ECs, we have also identified several novel EC-restricted genes, the function of which have yet to be fully defined. Conclusion: The study provides an initial catalogue of EC-restricted genes most of which are ubiquitously expressed in different endothelial cells.

Cytotoxic T lymphocytes (CTL) protect against viruses including HIV-1. To avoid viral escape mutants that thwart immunity, we chose 25 CTL epitopes defined in the context of natural infection with functional and/or structural constraints that maintain sequence conservation. By combining HLA binding predictions with knowledge concerning HLA allele frequencies, a metric estimating population protection coverage (PPC) was computed and epitope pools assembled. Strikingly, only a minority of immunocompetent HIV-1 infected individuals responds to pools with PPC >95%. In contrast, virus-naive individuals uniformly expand IFNγ producing cells and mount anti-HIV-1 cytolytic activity. This disparity suggests a vaccine design paradigm shift from infected to normal subjects.